Monoclonal antibodies

ABSTRACT

The present invention provides monoclonal antibodies against peptides having an amino acid sequence described in Sequence No. 1 or No. 2, the peptides being found in human thymidine phosphorylase and human platelet-derived endothelial cell growth factor. The Invention also provides an immunoassay for human thymidine phosphorylase and/or human platelet-derived endothelial cell growth factor using the monoclonal antibodies. The monoclonal antibodies of the invention recognize human thymidine phosphorylase and human platelet-derived endothelial cell growth factor, and thus are useful in the diagnosis and treatment of various tumors and their metastasis and diseases accompanying abnormal angiogenesis.

This application is a 371 of PCT/JP95/02661 filed Dec. 25, 1995.

TECHNICAL FIELD

The present invention relates to monoclonal antibodies which are usefulfor the detection of human thymidine phosphorylase and theplatelet-derived endothelial cell growth factor (PD-ECGF), i.e., for themonitoring via diagnosis and treatment of a variety of tumors, diseasesinvolving abnormal angiogenesis such as metastasis of tumors, rheumaticarthritis, diabetic retinitis, immature cataract, senile maculardegeneration, etc., as well as for use in drug delivery systems formedicine, etc.

TECHNICAL BACKGROUND

Thymidine phosphorylase is an enzyme essential to the metabolism ofthymidine and has been reported to manifest its activities in differenttissues (liver, lungs, small intestine, large intestine, placenta, etc.)of humans and animals J. Natl. Cancer Inst., 58, 1587-1590 (1977)!.Thymidine phosphorylase has also been reported to exhibit higher levelsof activity in various malignant tumors than in normal tissue Chem.Pharm. Bull., 34, 4225-4232 (1986)!. Because of this feature, thymidinephosphorylase is a target enzyme for an anticancer agent.

It is also reported that activity of PD-ECGF becomes elevated whenabnormal angiogenesis occurs as compared to normal tissue Nature, 338,557-562 (1989)!, which makes PD-ECGF a marker of diseases accompanyingabnormal angiogenesis.

Recently, human thymidine phosphorylase has been reported to begenetically identical to PD-ECGF Nature, 338, 557-562 (1989), Nature,356, 668 (1992), and J. biochem., 114, 9-14 (1993)!. Measurement ofthymidine phosphorylase activity and PD-ECGF activity is useful in thediagnosis of not only malignant tumors but also diseases involvingabnormal angiogenesis, such as rheumatic arthritis, diabetic retinitis,immature cataract, senile macular degeneration, etc.

Conventionally, activities of human thymidine phosphorylase and PD-ECGFhave been determined by measuring them in tissue. Therefore, excision ofnecessary amounts of tissue and fractionation of a crude enzymatic fluidmust be performed, calling for cumbersome handling techniques. Inaddition, detailed comparison on the intercellular level cannot be madeby conventional methods, raising a great problem in both the practicaland accuracy aspects.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide simple,widely-applicable, and practical means which enables specificmeasurement of thymidine phosphorylase/PD-ECGF.

The present inventors conducted careful studies, created monoclonalantibodies using as immunogens certain peptides in human thymidinephosphorylase and PD-ECGF protein, and found that they specificallyrecognize human thymidine phosphorylase and PD-ECGF and thus are usefulfor the diagnosis of various diseases accompanying elevated humanthymidine phosphorylase activity and PD-ECGF activity. The presentinvention was accomplished based on this finding.

Accordingly, the present invention provides a monoclonal antibodyagainst a peptide having an amino acid sequence described in SequenceNo. 1, a peptide having an amino acid sequence described in Sequence No.2, or against a peptide recognized by the presence of either one ofthese two peptides which serves as an antigen site.

The present invention also provides an immunoassay for human thymidinephosphorylase and/or PD-ECGF using the above monoclonal antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are a chart showing profiles of reaction specificity ofmonoclonal antibodies of the present invention.

FIG. 2 is a chart showing results of comparison in expression betweenhuman breast cancerous tissue which reacts with a monoclonal antibody ofthe present invention and its adjacent normal tissue (3 cases).

FIG. 3 is a photograph showing the result of immunohistochemicalstaining of a slice from healthy human large intestine tissue with whicha monoclonal antibody of the present invention reacts with specificity.

FIG. 4 is a photograph showing the result of immunohistochemicalstaining of a slice from human large intestinal cancerous tissue withwhich a monoclonal antibody of the present invention reacts withspecificity.

BEST MODE FOR CARRYING OUT THE INVENTION

The peptide having an amino acid sequence described in Sequence No. 1constitutes a portion of human thymidine phosphorylase, as does thepeptide having an amino acid sequence described in Sequence No. 2. Thepeptide recognized by the presence of either one of these two peptideswhich serves as an antigen site is not particularly limited so long asit contains an amino acid sequence described in Sequence No. 1 or 2.Examples of such a peptide include human thymidine phosphorylase,PD-ECGF, fragmentary peptides which are portions of human thymidinephosphorylase or PD-ECGF and which contain an amino acid sequencedescribed in Sequence No. 1 or 2, and peptides obtained by binding anyof these peptides to a carrier protein. Examples of the carrier proteininclude, but are not limited to, glutathione-s-transferase (GST),hemocyanine of Keyhole limpet hemocyanin (KLH), etc.

The monoclonal antibodies of the present invention may be prepared, forexample, by the cultivation of clones which produce antibodies thatrecognize as antigens the aforementioned peptides.

Such clones are obtained using a conventional cell fusion method; i.e.,forming fusion hybrids between antibody-producing cells and myelomacells, cloning the hybrids, and selecting clones producing antibodiesthat recognize as antigens the aforementioned peptides.

Examples of the antibody-producing cells used herein include spleencells, lymph node cells, and B lymphocytes, all of which are obtainedfrom animals immunized using, as antigens, peptides having the aminoacid sequence of Sequence No. 1 or 2, peptides recognized by thepresence of either one of these two peptides serving as an antigen site,or binding products of any of these peptides and a carrier protein.

The antibody-producing cells are prepared by routine methods. Forexample, an antigen is first emulsified in a complete or incompleteFreund's adjuvant to obtain an antigen suspension. Immediatelythereafter, the suspension is subcutaneously or intraperitoneallyinjected into an animal every 2-3 weeks for several times (preferably 3times), thereby immunizing the animal. Examples of animals which areimmunized include mice, rats, horses, goats, rabbits, etc. Three to fivedays after the final immunization performed via intravenousadministration of antigen, antigen-producing cells such as spleen cellsare harvested from the immunized animal.

As the myeloma cells, those derived from mice, rats, and humans areused. Preferably, myeloma cells derived from the same animal from whichthe antigen-producing cells are harvested are used. For example, as afusion counterpart of murine spleen cells, murine myeloma cells such asP3UI and SP-2/O-Ag 14 Nature, 277, 131-133 (1979)! may be used.

Fusion of cells is performed using a method described in Nature 256,495-497 (1975), or the method described by Ueda et al. in Proc. Natl.Acad. Sci. U.S.A 78, 5122-5126 (1981) or its equivalent method.Generally, 30-50% polyethylene glycol (average molecular weight:1000-4000) is used in a fusion reaction, and the reaction proceeds at30°-40° C. for 1-3 minutes. More preferably, 30-50% polyethylene glycol(average molecular weight: 4000) is used in a fusion reaction, and thereaction proceeds at 37° C. for 1-3 minutes.

The hybridoma obtained by cell fusion are subjected to culturing in, forexample, a microplate using an HAT medium (basal medium containinghypoxanthine 100 μM, aminopterin 0.4 μM, thymidine 16 μM) or a similarmedium, after which the grown cells are selected. The selectedhybridomas are subjected to an antigen-binding assay and then tocloning.

Briefly, antigen titer of the supernatant of the wells in which growthof cells was observed is determined by an enzyme antibody technique suchas ELISA (enzyme-linked immunosorbent assay, Miller, M. E., Lancet, 1,665 (1971)) using an antigen peptide as antigen for screening.Subsequently, cloning is performed using a limited dilution method,thereby obtaining clones.

When the thus-obtained clones are cultured like ordinary animal cells,the monoclonal antibodies of the present invention are produced in themedium. Since these clones, when intraperitoneally implanted in a Balb/cmouse to which 0.5 ml of pristan has been administered in advance,produce ascites containing high levels of monoclonal antibodies on day7-14, the monoclonal antibodies of the present invention can also becollected from ascites.

The monoclonal antibodies of the present invention can be recovered fromthe culture mixture or ascites containing the clones by the applicationof a well-known method for the purification of IgG, including a varietyof chromatography procedures using anion exchangers, hydroxyapatite, orprotein A- or G-immobilized columns; ammonium sulfate fractionationmethod; PEG fractionation method; ethanol fractionation method; or ahypotonic buffer precipitation method.

Among the thus-produced monoclonal antibodies of the present invention,those belonging to the class IgG are preferred.

Among the monoclonal antibodies of the present invention, those obtainedusing the entirety of human thymidine phosphorylase as antigen are lesspreferred than those obtained using as antigen a peptide which is aportion of human thymidine phosphorylase and which contains an aminoacid sequence of Sequence No. 1 or 2, or a binding product of such apeptide and a carrier.

The monoclonal antibodies obtained by the use of human thymidinephosphorylase fragments as antigen exhibit lower cross-reactivity toexogenous proteins than do antibodies against the entirety of humanthymidine phosphorylase. As a result, when antibodies against humanthymidine phosphorylase fragments are used in the diagnosis of cancers,higher sensitivity can be obtained than in the case where antibodiesagainst the entirety of human thymidine phosphorylase are used.Antibodies obtained using as antigen such human thymidine phosphorylasefragments are immunologically reactive with the entire human thymidinephosphorylase.

It is well known that each of the Fv, Fab, and F(ab')2 fragments of eachmonoclonal antibody of the present invention has a binding site for theantibody, and that these fragments have ability to bind antigens likecomplete antibodies. The fragments can be readily obtained viafragmentation of antibodies using a protease.

Diagnostic drugs making use of the monoclonal antibodies of the presentinvention may be combined with a variety of detection methods in whichsensitivity of measurement is attempted to be enhanced, for example,with the avidin-biotin-peroxydase complex (ABC) method in ELISA, thestreptoavidin-biotin complex (SBC) method, or theperoxydase-antiperoxydase (PAP) method, etc. The diagnostic drugs may beused singly or in combination with any one of other diagnostic drugs.

The monoclonal antibodies obtained in accordance with the presentinvention recognize with specificity human thymidine phosphorylase andPD-ECGF, and thus are useful in immunochemical and immunihistologicaldiagnosis of diseases involving promoted thymidine phosphorylase andPD-ECGF activities. Examples of such diseases include, but are notlimited to, malignant tumors and metastasis cancers resulting therefrom,rheumatic arthritis, diabetic retinitis, immature cataract, senilemacular degeneration, etc. Diagnosis may also be performed using bodyfluids, because the present antigens are transferred into body fluids ascell death occurs. Diagnostic drugs making use of the monoclonalantibodies of the present invention may be combined with a variety ofdetection methods in which measurement sensitivity is attempted to beelevated, including the ABC method in ELISA, the SBC method, or with thePAP method. The diagnostic drugs may be used singly or in combinationwith any one of other diagnostic drugs.

The monoclonal antibodies of the present invention may also be appliedto imaging of tissues, e.g., imaging diagnoses using indium-Ign-labelledantibodies.

Moreover, against malignant tumors, the monoclonal antibodies of thepresent invention can be used in targeting treatment using immunotoxinor the like in which antibody is bound to the active site of lysin orabrin, as reported in Cancer Res., 42, 457-464 (1982).

EXAMPLES

The present invention will next be described by way of examples.However, the present invention is not limited to these examples.

Example 1

Preparation of a binding protein between the N-terminal region or theC-terminal region of an antigen-human thymidine phosphorylase or PD-ECGFand glutathione transferase:

A fusion protein with GST was created using a commercially availablekit, GST-gene fusion system (Pharmacia). Briefly, integration into an E.coli expression vector was performed so as to obtain a fusion proteinbetween a polypeptide stretching from the 7th residue to the 250thresidue in the N-terminal of human thymidine phosphorylase/PD-ECGF andGST, or a fusion protein between a polypeptide stretching from the 353rdresidue to the 483rd residue in the C-terminal of human thymidinephosphorylase/PD-ECGF and GST.

The resultant plasmid was introduced into E. coli DH 5α, and cultured in200 ml of an LB medium liquid containing 100 μg/ml of ampicillin untilabsorbance at the wavelength of 660 nm reached 0.6. When absorbancereached 0.6, isopropyl-β-D(-)-thiogalactopyranoside (IPTG) was added.Culturing was continued for a further 3 hours, after which only E. colicells were collected by centrifugal separation. Collected cells werelysed in an SDS-polyacrylic amide sample buffer, and the solution wassubjected to fractional SDS-polyacrylic amide electrophoresis using adevice manufactured by Nippon Eido K.K. so as to fractionally obtain acorresponding protein. Yields were 6.4 mg of antigen for the N-terminalside, and 5.7 mg of antigen for the C-terminal side.

Example 2

Cloning of anti-human thymidine phosphorylase or PD-ECGF monoclonalantibody producing cells:

Three 8-week-old female Balb/c mice (supplied by Japan CLER K.K.) werefirst immunized with a binding protein prepared in Example 1, i.e., aprotein prepared by binding the N-terminal region or the C-terminalregion of thymidine phosphorylase or PD-ECGF to GST, the binding proteinbeing suspended in complete Freund's adjuvant. Subsequently, a 0.4 mlsolution containing 100 μg of a fusion protein was intraperitoneallyadministered to each mouse. On day 14 from the first immunization, afusion protein dissolved in a complete Freund's adjuvant was boosted.Further, as a final immunization, auxiliary immunization wasintraperitoneally performed on day 10 from the second immunization. Fourdays thereafter, the spleen of the mouse was removed. The spleen, aftergradually homogenized using 3 ml of a growth medium, was subjected tocentrifugal separation at 200 G to collect spleen cells.

The collected cells were slowly suspended in an RPMI 1640 medium (PEG400 solution) containing 50% (V/V) of polyethylene glycol (PEG), andgradually diluted with an RPMI 1640 medium to make the concentration ofPEG 5% (V/V). Cells were centrifugally separated. A growth medium wasgradually dispersed. Cells were seeded in wells of a microtiter plate inan amount of 10⁶ cells/0.1 ml per well. Cells were incubated in 5% CO₂at 37° C. for 2 minutes.

In this way, mouse spleen cells and myeloma cells P3U1 (P3X 63Ag8U.1)were fused using an electric fusing apparatus.

On the first day after cell fusion, 0.1 ml of an HAT medium was added.Thereafter, a half of the HAT medium was changed to a fresh HAT mediumevery 3 days. After 8 days, clones emerged. Before the elapse of 10days, the supernatant was subjected to a solid phase-antibody bindingtest (ELISA) for screening and checking of the production of IgG.

The positive groups in terms of antibody production were spread in aplate having 24 wells. When the cell density became high, the cells weretransferred to a 25 cm² culture. While maintaining hybridomas in an HTmedium (an HAT medium without containing aminopterin), antibodyproduction was checked.

In order to obtain clones that react with either the N-terminal orC-terminal of thymidine phosphorylase or PD-ECGF with specificity,screening was performed using an ELISA technique. Briefly, 50 μl of afusion polypeptide prepared by the use of GST as antigen was added toeach well of a 96-well microtiter plate designed for ELISA at aconcentration of 10 μg/ml, after which the wells were left overnight at4° C. Subsequently, 200 μl of a commercially available blocking liquid(product of Dainippon Pharmaceutical Co., Ltd.) was added to each well,and the wells were left at room temperature for 2 hours. A culturesupernatant of cloned hybridoma cells was added and incubated at 37° C.for 1 hour.

Peroxidase-labelled anti-mouse IgG antibody (product of OrganonTechnica) was added so as to achieve a final concentration of 1.0 μg/ml,followed by incubation at 37° C. for 1 hour. Subsequently, 100 μl of anOPDA/aqueous hydrogen peroxide solution was added, and reaction wasallowed to proceed for 20 minutes at room temperature. After completionof reaction, reaction was stopped by the addition of 100 μ1 of 2Nsulfuric acid. Absorbances at 490 nm and 595 nm were measured using amicrotiter reader. Screening was performed based on the differencebetween the obtained two absorbance data. The results are shown inTables 1 and 2.

                  TABLE 1                                                         ______________________________________                                        Clones resulted from the N-terminal peptide                                   Absorbance 490-595 nm                                                               (1) Immobilized with                                                                        (2) Immobilized with                                      Clone PD-ECGF/Thymidine                                                                           GST - C-terminal                                                                             (1)/(2) × 100                        No.   Phosphorylase Polypeptide    (%)                                        ______________________________________                                        1D10  0.720         0.990          72.7                                       1F4   0.700         0.875          80.0                                       2A4   0.794         0.941          84.4                                       2A11  0.779         0.936          83.2                                       2E6   0.747         0.929          80.4                                       3C9   0.805         0.996          80.8                                       ______________________________________                                    

Binding assay using respective clones, antigen, and PD-ECGF or thymidinephosphorylase

                  TABLE 2                                                         ______________________________________                                        Clones resulted from the C-terminal peptide                                   Absorbance 490-595 nm                                                               (1) Immobilized with                                                                        (2) Immobilized with                                      Clone PD-ECGF/Thymidine                                                                           GST - C-terminal                                                                             (1)/(2) × 100                        No.   Phosphorylase Polypeptide    (%)                                        ______________________________________                                        1F3   0.539         0.721          74.8                                       1F9   0.564         0.726          77.7                                       1H10  0.595         0.812          73.3                                       2C9   0.635         0.847          75.0                                       2D9   0.621         0.707          87.8                                       2H8   0.642         0.734          87.5                                       ______________________________________                                    

As a result, 6 strains were obtained which were positive for theN-terminal antigen, and 6 strains were obtained which were positive forthe C-terminal antigen.

Next, in order to investigate the binding ability of each clone tothymidine phosphorylase/PD-ECGF, screening was performed via ELISA,adding only 1 μg/ml of thymidine phosphorylase/PD-ECGF.

Clones were selected which bound themselves to a fusion protein betweenGST and the N-terminal thymidine phosphorylase/to PD-ECGF, and whichalso bound to thymidine phosphorylase/PD-ECGF, so as to obtain clonesthat bound themselves to both N- or C-terminal peptide and thymidinephosphorylase/PD-ECGF very well (N-terminal clone: 2A4, C-terminalclone: 2D9).

Example 3

Production of anti-thymidine phosphorylase/PD-ECGF monoclonal antibodythrough cell culturing:

Anti-thymidine phosphorylase antibody-producing hybridomas weresubcultured in a 75 cm² -flask for tissue culturing using an RPMI 1640medium containing 10% (V/V) fetal calf serum,100 mM pyruvic acid, and2-mercaptoethanol.

When antibody was purified, a serum-free medium, PUF-1 (product ofGibco) was used for culturing.

In this medium, hybridoma cells which propagated to 8×10⁵ cells/ml wererecovered through centrifugal separation at 200 G for 5 minutes.

The collected culture supernatant was concentrated to a volume of notmore than 10 ml using a PM 10 ultrafiltration membrane (product ofAmicon), followed by filtration through a membrane filter of 0.45 μm.

The thus-obtained crude fraction was combined with an equal amount of abinding buffer, mixed well, and allowed to pass through a protein Acolumn which had been equilibrated using a binding buffer in advance,thereby binding IgG to protein A. The bound product was washed using 10ml of a binding buffer, after which an IgG fraction was recovered byeluting the IgG in 3 ml of an elution buffer.

To 3 ml of the resultant fraction, 1 ml of 1M Tris HCl buffer (pH 9.0)was added immediately to return the pH in the vicinity of neutrality.Subsequently, dialysis was performed against phosphate-buffered saline(PBS) overnight, thereby obtaining monoclonal antibody IgG with a highpurity.

The subclasses of the obtained anti-thymidine phosphorylase antibodieswere determined via enzyme immunoassay using a commercially availablemouse monoclonal antibody isotyping kit (product of Amersham).

Subclasses of the clones are shown in Tables 3 and 4.

                  TABLE 3                                                         ______________________________________                                        N-terminal monoclonal antibody-producing clones                                      No. of Clones  Subclasses                                              ______________________________________                                               1D10           IgG1                                                           1F4            IgG2a                                                          2A4            IgG2b                                                          2A11           IgG2b                                                          2E6            IgG1                                                           3C9            IgG2a                                                   ______________________________________                                    

Immunogloblin subclasses of the clones

                  TABLE 4                                                         ______________________________________                                        C-terminal monoclonal antibody-producing clones                                      No. of Clones  Subclasses                                              ______________________________________                                               1F3            IgG2a                                                          1F9            IgG1                                                           1H10           IgG2b                                                          2C9            IgG2b                                                          2D9            IgG2b                                                          2H8            IgG3                                                    ______________________________________                                    

Example 4

Production of anti-thymidine phosphorylase/PD-ECGF monoclonal antibodyusing mouse ascites:

Hybridoma cells were washed once using a serum-free RPMI 1640 medium,and then resuspended in an RPMI 1640 medium at a concentration of 1×10⁷cells/ml.

Hybridoma cells in the number of 5×10⁶ were intraperitoneally injectedto each nude mouse, Balbc/nu-nu, and 2 weeks thereafter, monoclonalantibodies were harvested from the abdominal cavity of the mouse.

The collected ascites was subjected to centrifugal separation at 1500 Gfor 15 minutes, and then filtered through a membrane filter of 0.45 μm.

The thus-obtained crude fraction was combined with an equal amount of abinding buffer, mixed well, and allowed to pass through a protein Acolumn which had been equilibrated using a binding buffer in advance,thereby binding IgG to protein A. The bound product was washed using 10ml of a binding buffer, after which an IgG fraction was recovered byeluting the IgG in 3 ml of an elution buffer.

To 3 ml of the resultant fraction, 1 ml of 1M Tris HCl buffer (pH 9.0)was added immediately to return the pH in the vicinity of neutrality.Subsequently, dialysis was performed against phosphate-buffered saline(PBS) overnight, thereby obtaining monoclonal antibody IgG with a highpurity.

The subclasses of the obtained anti-thymidine phosphorylase antibodieswere determined via enzyme immunoassay using a commercially availablemouse monoclonal antibody isotyping kit (product of Amersham).

The subclasses of the clones were identical to those determined inExample 3.

Example 5

Detection of thymidine phosphorylase/PD-ECGF by immunoblotting:

200 μg of a crude protein extract were prepared using cells, normaltissue, or tumor tissue. The extract was analyzed by SDS-polyacrylamidegel electrophoresis using a gradient gel containing 4%-20% (W/V)acrylamide. Subsequently, the protein was electrically transcribed ontoa commercially available PVDF membrane (product of Millipore).

After non-specific adsorption was blocked using skim milk, theanti-thymidine phosphorylase/PD-ECGF antibody (clone 2A4) purified inExample 4 was added at a concentration of 0.5 μg/ml. The mixture wasallowed to stand at room temperature for 1 hour, and then at 4° C.overnight.

The peroxidase-labelled anti-mouse immunoglobulin used was acommercially available one. Color development of peroxydase wasperformed using an ECL system (Amersham). The results are shown in FIGS.1 and 2.

FIG. 1 shows the results of staining of N-terminal antibody 2A4 andC-terminal antibody 2D9, both prepared in Example 4. The fusion proteinbetween GST and the N-terminal polypeptide reacted with 2A4 withspecificity, and the fusion protein between GST and the C-terminalpolypeptide reacted with 2D9 with specificity. Both antibodies werereactive with over-expressed KB cells obtained by introducing CDNA ofthymidine phosphorylase/PD-ECGF, clearly showing a band at the positioncorresponding to a molecular mass of 55 kD.

FIG. 2 shows the results of comparison between human breast canceroustissue and its adjacent normal tissue (3 cases). As is understood fromthe bands at positions corresponding to a molecular mass of 55 kD,higher expression of thymidine phosphorylase/PD-ECGF was observed ineach cancerous tissue than in the normal tissue indicated on the right.

The lanes in FIG. 1 are as follows:

GST-N; a polypeptide in which GST was bound to the N-terminal peptide ofhuman thymidine phosphorylase,

GST-C; a polypeptide in which GST was bound to the C-terminal peptide ofhuman thymidine phosphorylase,

KB; a cell extract of KB cells, and

KB-TP; a cell extract of cells obtained by transfecting KB cells withcDNA of human thymidine phosphorylase/PD-ECGF.

Example 6

Immunohistochemical staining of thymidine phosphorylase/PD-ECGF inclinically obtained human tissue slices:

Human malignant tumor tissue and its adjacent normal tissue wereexcised, and immediately fixed using a 10% (V/V) formalin--PBS. Afterbeing left at room temperature for 24 hours, the fixed tissues wereembedded in paraffin, and slices each having a thickness of 3 μm wereprepared.

After the slices were deparaffinated, a pure thymidine phosphorylaseantibody (clone 2A4) purified in Example 4 was added thereto at aconcentration of 1 μg/ml, followed by standing at room temperature for 1hour, and then at 4° C. overnight. The slices were stained using acommercially available kit, Funakoshi ABC ellite kit (product ofVector). The results are shown in FIGS. 3 and 4.

FIG. 3 shows the negative result of staining of a slice from normaltissue. FIG. 4 shows the stained result of large intestinal canceroustissue. As is clearly understood from these figures, cytoplasm of tumortissue was positively stained. It was thus found that the antibody is avery useful one, being easy to use and suited in clinical applications,because its reactivity is not lost even after antigenity is degraded dueto paraffin fixing, or even after considerable time has elapsed aftertissue is excised.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 244 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ProGlyThrGlyAlaProProAlaProGlyAspPheSerGlyGluGly                              151015                                                                        SerGlnGlyLeuProAspProSerProGluProLysGlnLeuProGlu                              202530                                                                        LeuIleArgMetLysArgAspGlyGlyArgLeuSerGluAlaAspIle                              354045                                                                        ArgGlyPheValAlaAlaValValAsnGlySerAlaGlnGlyAlaGln                              505560                                                                        IleGlyAlaMetLeuMetAlaIleArgLeuArgGlyMetAspLeuGlu                              65707580                                                                      GluThrSerValLeuThrGlnAlaLeuAlaGlnSerGlyGlnGlnLeu                              859095                                                                        GluTrpProGluAlaTrpArgGlnGlnLeuValAspLysHisSerThr                              100105110                                                                     GlyGlyValGlyAspLysValSerLeuValLeuAlaProAlaLeuAla                              115120125                                                                     AlaCysGlyCysLysValProMetIleSerGlyArgGlyLeuGlyHis                              130135140                                                                     ThrGlyGlyThrLeuAspLysLeuGluSerIleProGlyPheAsnVal                              145150155160                                                                  IleGlnSerProGluGlnMetGlnValLeuLeuAspGlnAlaGlyCys                              165170175                                                                     CysIleValGlyGlnSerGluGlnLeuValProAlaAspGlyIleLeu                              180185190                                                                     ThrAlaAlaArgAspValThrAlaThrValAspSerLeuProLeuIle                              195200205                                                                     ThrAlaSerIleLeuSerLysLysLeuValGluGlyLeuSerAlaLeu                              210215220                                                                     ValValAspValLysPheGlyGlyAlaAlaValPheProAsnGlnGlu                              225230235240                                                                  GlnAlaArgGlu                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 130 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspProGlyLeuAlaArgAlaLeuCysSerGlySerProAlaGluArg                              151015                                                                        ArgGlnLeuLeuProArgAlaArgGluGlnGluGluLeuLeuAlaPro                              202530                                                                        AlaAspGlyThrValGluLeuValArgAlaLeuProLeuAlaLeuVal                              354045                                                                        LeuHisGluLeuGlyAlaGlyArgSerArgAlaGlyGluProLeuArg                              505560                                                                        LeuGlyValGlyAlaGluLeuLeuValAspValGlyGlnArgLeuArg                              65707580                                                                      ArgGlyThrProTrpLeuArgValHisArgAspGlyProAlaLeuSer                              859095                                                                        GlyProGlnSerArgAlaLeuGlnGluAlaLeuValLeuSerAspArg                              100105110                                                                     AlaProPheAlaAlaProSerProPheAlaGluLeuValLeuProPro                              115120125                                                                     GlnGln                                                                        130                                                                           __________________________________________________________________________

Hybridomas 2A4 and 2D9 described in the present application weredeposited at the National Institute of Bioscience and Human-TechnologyAgency of Industrial Science and Technology, 1-3, Higashi 1 chomeTsukuba-shi, Ibaraki-ken 305, JAPAN, on Oct. 14, 1997, under FERMBP-6142 and FERM BP-6143, respectively.

Industrial Utility

Since the monoclonal antibodies of the present invention enablemeasurement of thymidine phosphorylase/PD-ECGF with specificity andprovide a practical means which is simple and widely applicable, themonoclonal antibodies are useful for the diagnosis of a variety ofdiseases accompanying elevation in human thymidine phosphorylase orPD-ECGF activity.

We claim:
 1. A monoclonal antibody having binding specificity to apeptide having the amino acid sequence of SEQ ID NO:1, or an epitopethereof, wherein said monoclonal antibody is 2A4 produced by hybridomaFERM BP-6142, and is of the IgG_(2b) subclass.
 2. A monoclonal antibodyhaving binding specificity to a peptide having the amino acid sequenceof SEQ ID NO:2, or an epitope thereof, wherein said monoclonal antibodyis 2D9 produced by hybridoma FERM BP-6143, and is of the IgG_(2b)subclass.
 3. An immunoassay for detecting human thymidinephosphorylase/human platelet-derived endothelial cell growth factorcomprising the steps of:(A) reacting a body fluid or tissue sample witha monoclonal antibody having binding specificity to a peptide having theamino acid sequence of SEQ ID NO:1, or an epitope thereof, wherein saidmonoclonal antibody is 2A4 produced by hybridoma FERM BP-6142, and is ofthe IcG_(2b) subclass; and (B) determining whether said monoclonalantibody binds to proteins in said body fluid or tissue sample.
 4. Theimmunoassay as claimed in claim 3, wherein said immunoassay is animmunohistochemical staining assay.
 5. An immunoassay for detectinghuman thymidine phosphorylase/human platelet-derived endothelial cellgrowth factor comprising the steps of:(A) reacting a body fluid ortissue sample with a monoclonal antibody having binding specificity to apeptide having the amino acid sequence of SEQ ID NO:2, or an epitopethereof, wherein said monoclonal antibody is 2D9 produced by hybridomaFERM BP-6143, and is of the IaG_(2b) subclass; and (B) determiningwhether said monoclonal antibody binds to proteins in said body fluid ortissue sample.
 6. The immunoassay as claimed in claim 5, wherein saidimmunoassay is an immunohistochemical staining assay.